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. 2022 Feb 1;11:e74447. doi: 10.7554/eLife.74447

Figure 9. Smc3 acetylation is essential for meiosis.

(A–D) smc3-K112,113R leads to a global reduction in chromosomal Smc3 levels, which is only partially restored by WPL1 deletion. SMC3 (AM29315), smc3-K112,113R (AM29316), SMC3 wpl1Δ (AM30310), and smc3-K112,113R wpl1Δ (AM30311) strains carrying ndt80Δ were harvested 6 hr after induction of sporulation. (A) Flow cytometry profiles show similar DNA content at harvesting in all cultures. (B) Western immunoblot with Kar2 loading control (α-Kar2) shows comparable Smc3 (α-Smc3) and Rec8 (α-Rec8) levels in all cultures at the time of harvesting. (C) Calibrated Smc3 ChIP-seq for a representative region surrounding CEN10. (D) Mean calibrated ChIP-seq reads (line), standard error (dark shading), and 95% confidence interval (light shading) at all 16 centromeres, 32 borders, and 32 flanking arm sites. (E–G) Smc3-Ac is required to ensure co-segregation of sister chromatids in meiosis I and accurate meiosis II chromosome segregation. Meiotic progression (E) and meiosis I (F) and II (G) chromosome segregation were scored after live-cell imaging as in Figure 6 (B–E). Strains used were spo11Δ (AM30238), spo11Δ smc3-md (AM30240), spo11Δ SMC3 (AM30242), spo11 smc3-K112,113R (AM30244), spo11Δ wpl1Δ (AM30234), spo11Δ wpl1Δ smc3-md (AM30235), spo11Δ wpl1Δ SMC3 (AM30655), and spo11Δ wpl1Δ smc3-K112,113R (AM30237).

Figure 9—source data 1. Source images for the blot in Figure 9B.
Figure 9—source data 2. Source images for the blot in Figure 9B.
Figure 9—source data 3. Source images for the blot in Figure 9B.

Figure 9.

Figure 9—figure supplement 1. A system to express Smc3-K112,113R in meiosis.

Figure 9—figure supplement 1.

Ectopic expression of SMC3 and SMC3-K112,113R in meiosis. (A) Strategy to express non-acetylatable Smc3-K112,113R as the only Smc3 source in meiosis. In all diploid strains, both copies of endogenous SMC3 are placed under control of the CLB2 promoter, which is repressed in meiosis. Either wild-type SMC3 or smc3-K112,113R under the endogenous promoter are integrated at an ectopic locus of one parent (heterozygous). (B, C) Smc3-K112,113R is expressed but residual Smc3 persists from the smc3-md construct after meiotic induction. Wildtype (AM11633), smc3-md (AM28718), SMC3 (AM29315), and smc3-K112,113R (AM29316) carrying ndt80Δ were induced to sporulate. (B) Flow cytometry confirms DNA replication and prophase arrest. (C) Western immunoblot shows Smc3 (α-Smc3) and Kar2 (α-Kar2, loading control) levels at the indicated times after inducing sporulation. (D) Smc3 acetylation is required for meiosis. Spore viability of wild-type (AM25765), smc3-md (AM25067), SMC3 (AM25920), and smc3-K112,113R (AM28741) cells.
Figure 9—figure supplement 1—source data 1. Source images for the blot in Figure 9C.
Figure 9—figure supplement 1—source data 2. Source images for the blot in Figure 9C.
Figure 9—figure supplement 2. Establishment of meiotic cohesion requires Smc3 acetylation.

Figure 9—figure supplement 2.

Cells carrying ndt80Δ and heterozygous CEN5-GFP (A–C) or LYS2-GFP (D–F) were induced to sporulate. The mean percentage of cells with two visible CEN5-GFP (A) or LYS2-GFP (D) foci were scored at the indicated timepoints in four (A) or three (D) biological replicates (100 cells per timepoint). Bars show standard error. *p<0.05; **p<0.005, paired t-test. Western immunoblots (B, E) show Smc3 (α-Smc3) and loading control (α-Kar2) levels at the indicated timepoints for a representative experiment. (C, F) Flow cytometry shows DNA content at the indicated timepoints for a representative experiment. Strains used in (A–C) were wild-type (AM29184), smc3-md (AM29185), SMC3 (AM29186), or smc3-K112,113R (AM29187). Strains used in (D–F) were wild-type (AM29247), smc3-md (AM29248), SMC3 (AM29249), or smc3-K112,113R (AM29250).
Figure 9—figure supplement 2—source data 1. Source images for the blot in Figure 9B and E.
Figure 9—figure supplement 2—source data 2. Source images for the blot in Figure 9B and E.
Figure 9—figure supplement 3. Prevention of meiotic recombination allows smc3-K112,113R cells to efficiently exit prophase.

Figure 9—figure supplement 3.

Live-cell imaging of smc3-K112,113R cells demonstrates that defective meiosis I sister chromatid mono-orientation and meiosis II sister chromatid non-disjunction occur also in recombination-proficient cells that overcome the prophase delay, and that deletion of SPO11 allows cells to overcome this delay. Cells of the indicated genotypes were grown and analyzed as described in Figure 9B–E. (A) Meiotic progression was scored based on the number of Spc42-tdTomato foci. (B) Meiosis I chromosome segregation (heterozygous CENV-GFP) was scored in cells with two separated Spc42-tdTomato foci. (C) Meiosis II chromosome segregation (heterozygous CENV-GFP) was scored in cells with four separated Spc42-tdTomato foci. Strains used were AM29317 (wild type), AM29318 (smc3-md) AM29319 (SMC3), AM29320 (smc3-K112,113R), AM30238 (spo11Δ), AM30240 (spo11Δ smc3-md), AM30242 (spo11Δ SMC3), and AM30244 (spo11Δ smc3-K112,113R).